7. Discard the liquid, add PBS/Tween/milk (enough to immerse
the membrane), and incubate for 30 min at room temperature
with gentle oscillation. Add anti-GFP antibody and incubate
(see Note 13).
8. Discard the antibody solution and wash the membrane 3–4
times for 5 min in PBS/Tween.
9. Add PBS/Tween/milk containing the appropriate secondary
antibody conjugate (see Note 14) and incubate for 1 h with
gentle oscillation at room temperature.
10. Discard the antibody solution and wash the membrane 3–4
times for 5 min in PBS/Tween.
11. Remove the membrane from the box, briefly allow the solution
to drip off, and place it into a transparent plastic pouch (see
Note 9).
12. Add ECL substrate to the membrane and expose the chemiluminescent signal to film or appropriate imaging equipment
(Fig. 4a).
13. After imaging, the membrane should be incubated overnight
with Coomassie staining buffer with gentle oscillation, then
washed twice with destaining buffer (1 min per wash), rinsed
well with water, and allowed to dry. The blue staining allows to
assess the total protein content in each lane (Fig. 4b).
If the result of the western blot confirms the reproducibility of
the IPed protein samples (as in Fig. 4), these can be sent for mass
spectrometry and subsequent statistical analysis. Please refer to
ref. 8 for an example of methods used. However, we highly recommend to outsource this phase of the experiment to specialists.
3.6 Subcellular
Localization
of dsRNA-Associated
Protein Candidates
Following the identification of A. thaliana proteins isolated
together with dsRNA by mass spectroscopy, it is important to verify
their localization relative to virus replication complexes in vivo. To
achieve this, the proteins of interest are genetically fused with a red
fluorescent protein and transiently expressed in N. benthamiana
plants stably and ubiquitously expressing B2:GFP [7], the same
protein used in A. thaliana to pull down dsRNA (see Subheading
3.1). In the absence of viral infection, B2:GFP shows a diffuse
nucleocytoplasmic subcellular localization. However, upon infection with a variety of RNA viruses the localization of B2:GFP
changes dramatically, concentrating in bright and clearly visible
cytoplasmic foci [7, 8], which correspond to the dsRNAcontaining virus replication complexes. Transient expression of
the RFP-tagged dsRNA-binding protein candidates in these plants
and analysis by laser confocal microscopy allows to determine their
localization relative to the B2:GFP-labeled viral replication complexes (Fig. 5).
322
Marco Incarbone and Christophe Ritzenthaler
the membrane), and incubate for 30 min at room temperature
with gentle oscillation. Add anti-GFP antibody and incubate
(see Note 13).
8. Discard the antibody solution and wash the membrane 3–4
times for 5 min in PBS/Tween.
9. Add PBS/Tween/milk containing the appropriate secondary
antibody conjugate (see Note 14) and incubate for 1 h with
gentle oscillation at room temperature.
10. Discard the antibody solution and wash the membrane 3–4
times for 5 min in PBS/Tween.
11. Remove the membrane from the box, briefly allow the solution
to drip off, and place it into a transparent plastic pouch (see
Note 9).
12. Add ECL substrate to the membrane and expose the chemiluminescent signal to film or appropriate imaging equipment
(Fig. 4a).
13. After imaging, the membrane should be incubated overnight
with Coomassie staining buffer with gentle oscillation, then
washed twice with destaining buffer (1 min per wash), rinsed
well with water, and allowed to dry. The blue staining allows to
assess the total protein content in each lane (Fig. 4b).
If the result of the western blot confirms the reproducibility of
the IPed protein samples (as in Fig. 4), these can be sent for mass
spectrometry and subsequent statistical analysis. Please refer to
ref. 8 for an example of methods used. However, we highly recommend to outsource this phase of the experiment to specialists.
3.6 Subcellular
Localization
of dsRNA-Associated
Protein Candidates
Following the identification of A. thaliana proteins isolated
together with dsRNA by mass spectroscopy, it is important to verify
their localization relative to virus replication complexes in vivo. To
achieve this, the proteins of interest are genetically fused with a red
fluorescent protein and transiently expressed in N. benthamiana
plants stably and ubiquitously expressing B2:GFP [7], the same
protein used in A. thaliana to pull down dsRNA (see Subheading
3.1). In the absence of viral infection, B2:GFP shows a diffuse
nucleocytoplasmic subcellular localization. However, upon infection with a variety of RNA viruses the localization of B2:GFP
changes dramatically, concentrating in bright and clearly visible
cytoplasmic foci [7, 8], which correspond to the dsRNAcontaining virus replication complexes. Transient expression of
the RFP-tagged dsRNA-binding protein candidates in these plants
and analysis by laser confocal microscopy allows to determine their
localization relative to the B2:GFP-labeled viral replication complexes (Fig. 5).
322
Marco Incarbone and Christophe Ritzenthaler